Fluoride can be both beneficial and toxic. For years fluoride has been used in oral hygiene for its beneficial effects in products such as toothpaste and mouthwash. On the other hand, a build up of fluoride in a cell can be toxic. Therefore, appropriate amounts of fluoride and careful regulation of fluoride in cells is essential.
A key biochemical component of a bacterial cell's fluoride surveillance and response system is the new-found fluoride-responsive riboswitch class based on the crcB motif (Weinberg et al. 2010. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaeal, and their metagenomes. Genome Biol 11:R31). The general architectures and functional mechanisms for fluoride riboswitches are similar to many other known riboswitch classes. Members of each riboswitch class carry at least one ligand-binding “aptamer” domain and one adjoining “expression platform” domain that together control expression of the downstream gene(s) by one of several known mechanisms (Wickiser et al. 2005. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49-60; Barrick and Breaker, 2007. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol 8:R239). The most common mechanisms used by bacteria include transcription termination and translation initiation, although some bacterial riboswitch classes exploit other mechanisms such as allosteric ribozyme-mediated splicing (Lee et al. 2010. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329:845-848) and coenzyme-dependent ribozyme self-cleavage (Winkler W C, et al. 2004. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428:281-286).
Ligands for these riboswitches include fundamental metabolites such as coenzymes, amino acids, nucleobases and their derivatives, and amino sugars (Roth A, Breaker R R. 2009. The structural and functional diversity of metabolite-binding riboswitches. Annu Rev Biochem 78:305-334; Breaker R R. 2011. Riboswitches and the RNA World. In: RNA Worlds. J F Atkins, R F Gesteland, T R Cech, eds. CSH Press). Moreover, riboswitches with striking biochemical properties [e.g. ribozyme-based gene regulation (Lee E R, et al. 2010. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329:845-848; Winkler W C, et al. 2004. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428:281-286); digital regulation by either cooperative ligand binding (Mandal M, et al. 2004. A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science 306:275-279) or by tandem riboswitch function (Welz R, Breaker R R. 2007. Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis. RNA 13:573-582); two-input logic gates (Sudarsan N, et al. 2006. Tandem riboswitch architectures exhibit complex gene control functions. Science 314:300-304)] have been discovered as well as others that expose critical biological circuitry [e.g. bacterial second messenger sensing (Sudarsan N, et al. 2008. Riboswitches in eubacterial sense the second messenger cyclic di-GMP. Science 321:411-413; Lee E R, et al. 2010. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329:845-848); coenzyme-mediated alternative splicing control in eukaryotes (Cheah et al. 2007. Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature 447:497-501; Wachter et al. 2007. Riboswitch control of gene expression in plants by splicing and alternative 3′ end processing of mRNAs. Plant Cell 19:3437-3450)]. Furthermore, the first datasets that validate riboswitches as targets for antibacterial and antifungal agents have been provided (Sudarsan et al. 2005. Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. Chem Biol 12:1325-1335; Blount K F, et al. 2007. Antibacterial lysine analogs that target lysine riboswitches. Nature Chem Biol 3:44-49; Kim J N, et al. 2009. Design and antimicrobial action of purine analogues that bind guanine riboswitches. ACS Chem Biol 4:915-927; Lee E R, et al. 2009. Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression. RNA Biol 6:187-194; Blount K F, Breaker R R. 2007. Riboswitches as antibacterial drug targets. Nature Biotechnol 24:1558-1564). However, the identification of a fluoride-responsive riboswitch ranks as one of the most intriguing discoveries among this collection of riboswitch science advances.
Riboswitches are structured RNA domains commonly located in the 5′ untranslated regions (UTRs) of messenger RNAs where they selectively bind target metabolites (or ions in rare instances) and regulate gene expression (Mandal M, Breaker R R. 2004. Gene regulation by riboswitches. Nature Rev Mol Cell Biol 5:451-463; Roth A, Breaker R R. 2009. The structural and functional diversity of metabolite-binding riboswitches. Annu Rev Biochem 78:305-334; Smith A M, et al. 2010. Riboswitch RNAs: regulation of gene expression by direct monitoring of a physiological signal. RNA Biol 7:104-110). Recently a natural fluoride-responsive riboswitch class was discovered that offers an unprecedented opportunity to establish the molecular basis for fluoride sensing in organisms distributed among two of the three domains of life. Also, for the first time, strategies for fluoride toxicity alleviation in many species can be revealed.
The discovery of fluoride-responsive riboswitches exposes a widespread sensor and toxic response system for fluoride—an anion whose mechanisms of toxicity in bacteria and mechanisms of efficacy in humans remain incompletely defined. Reporter systems can be developed that detect the in vivo concentration of this toxic ion. Furthermore, such reporters can be used to identify compounds that influence fluoride uptake and retention, which has great potential for basic research and therapeutics development.
The discovery has immediate implications for understanding the health effects of fluoride additives in water and dental hygiene products. These studies involve the creation and exploitation of tools to sense and manipulate cellular fluoride concentrations. Systems that report the in vivo concentration of fluoride for high throughput (HTP) screening use and create compounds that manipulate these fluoride levels are disclosed. Of particular interest is the identification and optimization of compounds that can be used to sensitize cells to the effects of fluoride via fluoride transporter blocking, thus enhancing the antimicrobial action of this anion.
The disclosed invention provides compositions and methods for demonstrating compatibility of a riboswitch-reporter fusion system that measures cytoplasm fluoride concentrations and permits HTP screening for fluoride transporter inhibitors.
The disclosed invention further provides compositions and methods for using a riboswitch-reporter fusion system in HTP screens to identify compounds that increase fluoride concentrations in bacteria. Fluoride transporter inhibitors or fluoride uptake/diffusion facilitators can be identified by measuring the increase in reporter activity under otherwise non-permissive fluoride concentrations in media.
The disclosed invention further provides compounds and methods for establishing the mechanisms of various compounds which allow for the classification of targets for the future development of fluoride concentration agonists.
The disclosed invention further provides compounds and methods for determining the pharmacophore of fluoride transporter inhibitors and other fluoride agonist compounds. This can yield insight into the critical substructures of the active compounds and permit the synthesis and analysis of derivative compounds that can have improved efficacy.
The disclosed invention provides compositions and methods to identify novel riboswitch-targeting compounds in Gram positive organisms. The methods used to identify, characterize and enhance compounds that influence fluoride concentrations in cells can also be used to discover compounds that inhibit fluoride riboswitches other riboswitch classes. Such compounds could become leads for novel antimicrobial agents.
The disclosed invention further provides fluoride binding aptamers that can be used alone or in combination with an expression platform domain.